The FDA and blood banking societies recognize that bacterial detection to reveal contamination in stored blood products is a direct approach for reducing the problem of post-transfusion bacterial sepsis, and potentially can allow extension of currently approved product storage times. This project involves development of an in situ method (analysis completed within the sealed storage bag) for detecting the presence of likely bacterial contaminants by means of optical analysis at the blood bank or transfusion service immediately prior to release for patient use. Detection is through sensitive fluorescence technologies in which specialized biosensor molecules, immobilized on beads entrapped behind a porous barrier and maintained in continuous contact with the blood product, are interrogated for bacterial presence via non-invasive measurement of fluorescence in a bag-attached detection chamber through a fiber optic probe fluorometer. This methodology is designed to permit permanent association of the detector with the product bag, thereby allowing a real-time estimate of the contamination level in each unit. The project builds upon well established biological (bacteria secrete proteases) and chemical (such proteases are easily detected through "bead" techniques) facts, greatly raising probability of success for this unique application. Unique collaborative efforts with Los Alamos National Laboratories plus world-class consultants enhance the strengths of the applicant organization. Three specific aims are proposed for the current effort. First, modifications are being introduced to the detection chamber containing the biosensor beads to improve detection sensitivity and reduce background signal; additionally the fiber optic probe will be altered to accept an annular magnet for improved bead collection prior to measurement. Further tests of bead surface chemistries are proposed to improve bacterial adherence and concentrate them in the detector chamber. Second, the presence of proteases secreted by three types of bacteria known to contaminate platelets will be used as a test system for this method of analysis. This involves relating bacteria levels to secreted protease action which is observed by altered fluorescent properties of bead-bound substrates. The goal is detection of bacteria present in levels of 10(3) per mL or less. Third, the detection capability will be extended to additional protease-secreting bacteria that have been reported as platelet contaminants by analyzing their protease production during growth and by developing substrates more suitable for proteolytic action in this broader array of organisms. A plan for commercialization is well advanced, assuring that successful technical developments will rapidly move toward introduction into the industry.